Perfusion of the brain depends on the caliber of cerebral blood vessels. This proposal focuses on the regulation of cerebral artery diameter by intravascular pressure, adenosine, calcitonin gene-related peptide (CGRP) and nitric oxide through modulation of potassium channels (ATP-sensitive K+ (KATP) channels and calcium-activated K+ (Kca) channels). We have found that the important vasodilators, CGRP and adenosine, act in part through stimulation of KATP channels in smooth muscle, and in the case of CGRP, may involve activation of protein kinase A (PKA). Our previous publications and preliminary data form the basis of the first Specific Aim:
Aim 1 will address the key issue of how CGRP and adenosine signal KATP channels to open, i.e. the signal transduction pathways in these processes will be elucidated. Further, our preliminary data suggest that vasoconstrictors can inhibit KATP channels through multiple pathways including activation of protein kinase C. We will, therefore examine the mechanisms by which vasoconstrictors inhibit KATP channels, particularly after activation by CGRP and adenosine. Another issue of fundamental and paramount importance in the regulation of arterial tone is how smooth muscle membrane potential and diameter are regulated when pressure changes. Elevation of intravascular pressure causes a graded membrane depolarization and constriction (tone) of small arteries. We have found that this pressure-induced constriction is accompanied by activation of Kca channels which opposes the membrane depolarization to pressure, and thereby limits vasoconstriction. Recently, we have made a major advance in understanding this process by visualizing localized, ryanodine-sensitive bursts of Ca2+ (""""""""Ca2+ sparks"""""""") originating from the sarcoplasmic reticulum (SR) just under the surface membrane of smooth muscle cells from small (10 micron diameter) cerebral arteries. These Ca2+ sparks appear to arise from the opening of ryanodine-sensitive SR Ca2+ release channels. Therefore, in Aim 2, we will explore the exciting possibility that Ca2+ sparks are elementary events signaling Kca channels to open to control the membrane potential and diameter of small arteries. Our discovery of Ca2+ sparks in smooth muscle and that nitric oxide activates Kca channels through stimulation of cGMP-dependent protein kinase (PKG), suggested the possibility, which we will explore in Aim 3, that vasodilator (adenosine, CGRP, and nitric oxide) act also by stimulation of Kca channels through direct PKA and PK pathways, and indirectly through stimulation of Ca2+ sparks. Indeed, vasodilator activation of Ca2+ sparks could represent an important new mechanism of vasodilation. To accomplish the goals of the proposed project, we will use a unique combination of techniques including the measurements of arterial diameter, membrane potential Ca2+ sparks using laser scanning confocal microscopy, Ca2+ imaging, and K+ channel currents. The proposed study should provide major new insights into the control of arterial tone and reactivity by intravascular pressure vasodilators, and vasoconstrictors through regulation of K+ channels. Finally, the proposed project should suggest new strategies for treatment of vascular disorders such as hypertension, stroke, cerebral vasospasm and migraine.
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